UNIT 8.1
Maternal serum -fetoprotein (AFP) testing has been used for many years to screen for
open neural tube defects in the fetus (Wald and
Cuckle, 1979). Low levels of AFP have been
found to be associated with an increased risk of
Down syndrome as well (Cuckle et al., 1984;
Merkatz et al., 1984; DiMaio et al., 1987).
In addition, testing of maternal serum levels of unconjugated estriol (UE3) and human
Clinical
Cytogenetics
8.1.1
Supplement 89
is low. Trisomy 18 is also associated with abnormal values of these tests (low AFP, UE3,
and hCG), although the a priori likelihood of
trisomy 18 is lower than that of trisomy 21.
Overview of
Clinical
Cytogenetics
common obstetrical practice to discuss prenatal diagnostic options with pregnant women
aged 35 years or older. The existence of a
weak paternal age effect on the incidence of
trisomy has been suggested, but supportive evidence is less convincing (Hook, 1987; Stene
and Stengel-Rutkowski, 1987).
Family history
A number of factors in a family history may
indicate increased risk of a fetal chromosome
abnormality. For example, one parent may
have a balanced translocation. Such rearrangements are usually ascertained in the parent of a
child with an unbalanced karyotype and multiple congenital anomalies. A balanced translocation carrier is at increased risk of producing
unbalanced gametes and having a child with
congenital anomalies (Daniel et al., 1989). The
risk differs depending on the rearrangement,
but prenatal detection of such a chromosome
rearrangement is usually straightforward.
It is important to be alert to the possibility
of a chromosome rearrangement if there is a
family history of a child with a chromosome
anomaly. This applies even if the propositus
has Down syndrome, as a small proportion of
individuals with Down syndrome have translocation of chromosome 21 to another chromosome. If a couple seeks counseling and a relative has Down syndrome, records should be
sought to determine whether the Down syndrome was due to free trisomy or translocation. If such information cannot be obtained,
chromosomal analysis should be done on the
parent whose relative is affected to rule out
a balanced translocation. A couple who has a
child with trisomy has an empiric risk of recurrence of 1% (Lister and Frota-Pessoa, 1980).
This risk exceeds the risk of amniocentesis,
so such couples should be offered prenatal
testing.
Recurrent miscarriage
Balanced chromosome rearrangements can
lead to the production of gametes with unbalanced karyotypes. Most embryos with unbalanced karyotypes are nonviable and are spontaneously aborted during the first trimester
(Boue et al., 1973). A history of recurrent early
pregnancy loss may indicate the presence of a
balanced rearrangement in one of the parents.
Miscarriage is relatively common in the general population, so one or two miscarriages do
not indicate a high likelihood of a balanced
rearrangement. In couples with two or more
spontaneous abortions, the frequency of chromosome anomalies in one parent is 4% to 5%
8.1.2
Supplement 89
MOLECULAR CYTOGENETIC
METHODS
Fluorescence In Situ Hybridization
(FISH)
Fluorescence in situ hybridization (FISH;
Knoll and Lichter, 2005) is routinely
employed in the clinical cytogenetics laboratory. Probes for specific DNA sequences are
used to identify specific chromosome regions,
and are also used to detect microdeletions.
A number of microdeletion syndromes have
been identified. These syndromes are defined
clinically, and FISH studies with appropriate
probes can be used to detect the deletions for
UNIT 4.3,
Clinical
Cytogenetics
8.1.3
Current Protocols in Human Genetics
Supplement 89
Overview of
Clinical
Cytogenetics
8.1.4
Supplement 89
threshold with whole-chromosome aneuploidies, and with SNP-based microarrays. However, CMA is unable to detect truly balanced
chromosomal rearrangements, or low-level
mosaicism, as only robust copy-number information is provided (Miller et al., 2010; Bi et al.,
2013). Repetitive regions of the genome not
represented on the array (pericentromeric regions, heterochromatin) are also not analyzed.
Because CMA analysis is not able to determine
the structural consequences of copy-number
changes, in circumstances with low-level mosaicism, complex structural rearrangements,
and balanced translocations, G-banded karyotype and FISH are indicated (Miller et al.,
2010; Bi et al., 2013).
TISSUE SOURCES
Prenatal Analysis
Chorionic villus sampling
This procedure is usually performed at 10
to 12 weeks gestation. Fetal tissue is obtained
by insertion of a biopsy catheter either through
the cervix or transabdominally. The latter approach may be associated with a lower rate
of infectious complications. The fetal sample must be visually inspected and maternal
tissue dissected away prior to analysis. Cytogenetic studies can be performed on spontaneously dividing trophoblast cells or mesenchymal cells grown in culture (UNIT 8.3, Breman and Patel, 2012). The former permits very
rapid turnaround (as fast as 24 to 48 hr) but carries a risk of finding chromosomal anomalies
in some cells that may not be present in the fetus (Sachs et al., 1990). Such mosaicism is less
common in cultured chorionic villus cells and
still less so in amniotic fluid cells. Findings
from direct trophoblast analysis should be followed up with a study of cultured cells. Chorionic villus tissue can also be used as a source
of fetal DNA for molecular genetic studies.
The major advantage to chorionic villus
sampling is the ability to obtain fetal tissue
early in pregnancy. If termination of pregnancy is contemplated, the earlier termination
procedure is less traumatic and allows the patient to maintain privacy about the pregnancy.
Procedure-related complications, however, are
higher than with amniocentesis (1% compared with 0.5% for amniocentesis; Rhoads
et al., 1989). Concern has been raised about
distal limb reduction defects and anomalies of
the lower jaw and tongue in children born from
pregnancies studied by chorionic villus sampling (CVS; Burton et al., 1992; Schloo et al.,
Amniocentesis
Amniocentesis is usually performed between 15 and 18 weeks of gestation, and not
recommended before 15 weeks as the risk of
talipes equinovarus in the fetus is significant
(Tabor and Alfirevic, 2010). The rate of complications is comparable to CVS, at 0.5% to
1%. Fetal tissue obtained by this means must
be grown in culture for cytogenetic analysis,
which usually requires 1 to 2 weeks (UNIT
8.4, Miron, 2012). Two approaches to amniotic fluid cell culture are in wide use. Some
laboratories grow cells at low density on coverslips for several days, allowing individual cells
to grow into colonies, and then prepare cells
for analysis directly on these slips. Analysis
of cells in multiple colonies is helpful in distinguishing chromosome anomalies that arise
during the culturing process from those that
occur in the fetus. If all cells from all colonies
are abnormal, the fetus is likely to be abnormal. If all cells from two or more colonies
are abnormal, but other colonies are normal, it
is likely that the fetus is mosaic with a normal
and an abnormal cell line. If only one colony is
abnormal, it is possible that the fetus has lowlevel mosaicism or that a single cell may have
acquired an abnormality in vitro. If only some
cells of a single colony are abnormal, the mosaicism (referred to as pseudomosaicism) is
likely to have arisen in vitro (Wilson et al.,
1989).
The alternative culture system is to grow
cells in culture flasks. In this case, analysis of
individual colonies is not possible, and evidence of true fetal mosaicism will consist of
finding the same abnormality in cells from two
or more independent cultures.
Rapid prenatal cytogenetic diagnosis can
be achieved with FISH (ACMG, 2000; UNIT
4.3, Knoll and Lichter, 2005). Chromosomespecific cloned DNA sequences are fluorescently labeled and hybridized to interphase nuclei. A discrete fluorescent spot in each nucleus
should be observed representing each chromosome for which the probe is hybridized;
therefore trisomy results in three spots and
monosomy in only one spot. This approach
offers very rapid turnaround time (24 to 48
hr) but has some limitations. Clinical use has
focused only on detecting trisomy or monosomy for a few specific chromosomes: X, Y,
13, 18, and 21. Chromosome rearrangements,
Clinical
Cytogenetics
8.1.5
Current Protocols in Human Genetics
Supplement 89
Table 8.1.1 ACMG Recommendations for Cytogenetic Analysis of Pre- and Postnatal Samplesa
Tissue
Count
Analyze
Karyotype
20 cells from 2
cultures
5 cells
Chorionic villus
(culture plus direct)
5 cells
Constitutional (e.g.,
lymphocyte, fibroblast)
20 cells (30 if
sex-chromosome
abnormality is
suspected)
5 cells
a Source:
b For
ACMG (2009).
in situ studies, if 15 cells are not available, 10 must be used.
Postnatal Analysis
For CMA studies, genomic DNA can be extracted from the prior cell types and utilized for
copy-number analysis (UNIT 8.12, Miller et al.,
2012).
Fibroblasts
Overview of
Clinical
Cytogenetics
Fibroblasts obtained by skin or other tissue biopsy can be grown in culture and used
as a source of dividing cells for chromosome
analysis (UNIT 8.5, Levy et al., 2009). This approach is used in cases where peripheral blood
may be impossible to obtain (such as post-
Analytical Considerations
Number of cells
Recommendations of the American College of Medical Genetics and Genomics
(ACMG) are presented in Table 8.1.1. Counts
are defined as the number of centric chromosomes per metaphase cell. Analyzed cells are
banded chromosomes evaluated either through
the microscope or on digitized images or
photographs.
Cell counting should include noting any numerical and structural aberrations seen. Cells
that are fully karyotyped may be included in
8.1.6
Supplement 89
Staining methods
Different laboratories employ different routine staining methods (see UNIT 4.2, Schreck
and Dist`eche, 1994; and Benn and Perle, 1992,
for staining protocols; abbreviations defined in
the references are used below). GTG-banding
is the most commonly employed, although
some laboratories use QFQ- or R-banding.
Laboratories also must set a standard level of
resolution for routine analysis. Level of resolution is usually expressed in terms of banding
levelthat is, an estimate of the total number of bands visible in the entire haploid chromosome complement (Josifek et al., 1991). A
standard level of resolution for routine analysis is 400 to 550 bands. Identification of
some microdeletion syndromes requires substantially higher resolution, e.g., 800 bands.
High-resolution analysis can be accomplished
in a variety of ways. Usually extended chromosomes are prepared by synchronization
of cells and harvesting at prometaphase or
treatment with an intercalating agent such as
ethidium bromide.
Special stains are used to aid in the identification of chromosome rearrangements. These
techniques include C-banding, NOR-staining,
T-banding, replication banding, and sister
chromatid differentiation. Recently, FISH has
been added to the armamentarium for precise
chromosome identification (UNIT 4.3, Knoll and
Lichter, 2005). Specific DNA probes can help
to identify a rearranged chromosome or reveal
submicroscopic deletions.
Image capture
Images of chromosomes were traditionally
recorded by photomicrography, but the use of
computers has greatly modernized cytogenetic
analysis. Chromosome analysis may be done
through the microscope or by the use of digital images with karyotyping analysis software
on desktop computers. A number of digitalimage-capture devices are available (UNIT 4.4,
McNamara et al., 2005). These devices, which
can substantially speed the counting and analysis of chromosomes and provide a digital photograph of chromosomes, are currently in use
in most cytogenetic laboratories.
Reporting
Reports of cytogenetic results usually include at least the following elements:
CHROMOSOME ANOMALIES
Introduction
This section will describe major types of
chromosome anomalies. Detailed guides to
chromosome nomenclature can be found in
APPENDIX 4C and ISCN (2013). Specific syndromes will not be covered here; several
books describe the clinical features of major chromosomal anomalies (de Grouchy and
Turleau, 1984; Schinzel, 2001; Borgaonkar,
2011; Gersen and Keagle, 2013).
Numerical Abnormalities
Polyploidy
Tetraploidy occurs rarely as a constitutional
anomaly, probably because of early embryonic
lethality. Triploidy is common in early spontaneous miscarriages but may also be compatible with live birth. Triploid fetuses have the
karyotype 69,XXX or 69,XXY, or 69,XYY. A
69,YYY conceptus lacking an X chromosome
is nonviable. Some liveborn triploids are mosaics with a triploid and a diploid cell line, e.g.,
46,XX/69,XXX.
Clinical
Cytogenetics
8.1.7
Current Protocols in Human Genetics
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pericentric inversion
paracentric inversion
duplication
terminal
deletion
interstitial
deletion
isochromosome
ring chromosome
Trisomy
All possible chromosome trisomies have
been seen in the products of spontaneous abortions, but only a few (trisomies 8, 13, 18, 21,
X, and Y) occur with appreciable frequency
in liveborns. Trisomy is assumed to occur
by nondisjunction during meiosis or mitosis.
A trisomic karyotype is indicated using the
form 47,XY,+21 (male with trisomy 21). Sexchromosome aneuploidy is denoted 47,XXX
(triple X syndrome) or 47,XXY (Klinefelter
syndrome).
Monosomy
Overview of
Clinical
Cytogenetics
Structural Abnormalities
Chromosome structure abnormalities include a wide variety of different forms, as described below and depicted in Figure 8.1.1.
Deletion
Deletions can be either terminal or interstitial. Interstitial deletions involve two breaks
in the chromosome, loss of material, and reunion of the broken ends. Most deletions are
probably interstitial as all chromosome ends
must contain a telomere. The clinical effects
of deletion are due to monosomy for the
deleted chromosome material; or nullisomy,
8.1.8
Supplement 89
Duplication
In these abnormalities a segment of chromosome material is duplicated, usually in tandem with the original sequence. The duplicated region may have the same orientation as
the original sequence (direct duplication) or
may be repositioned by 180 (inverted duplication). The phenotype is due to trisomy for
the duplicated region.
Inversion
Here a segment of chromosome material is
inverted 180 with respect to its normal orientation. This involves two sites of breakage and
subsequent reunion. If the inversion includes
the centromere it is referred to as pericentric; otherwise it is paracentric. Inversions
may be associated with a clinical phenotype if
the site of breakage occurs in a gene leading
to disruption of the gene sequence or dysregulation of a gene(s), but often do not result in
an abnormal phenotype. Inversions can however lead to unbalanced recombinant chromosomes. During meiosis, pairing between homologous chromosomes in which one is inverted leads to formation of an inversion loop.
If a cross-over occurs within the loop, the
products contain duplications of some chromosome regions and deletions of others. In a
paracentric inversion, cross-overs result in dicentric or acentric fragments that tend to be
unstable at mitosis. The consequences of having an inversion therefore can be formation of
chromosomally unbalanced gametes, leading
to recurrent miscarriage or in some instances
the birth of offspring with congenital anomalies. Pericentric inversion of chromosome 9 is
a common heteromorphic variant of no known
clinical significance (Lubs et al., 1977). Another common pericentric inversion involves
chromosome 2 and is likewise not usually associated with an adverse outcome (MacDonald
and Cox, 1985).
Isochromosome
Isochromosomes contain two copies of
short-arm or long-arm material separated by
a centromere and are thought to arise by misdivision of the centromere. They result in
trisomy for the duplicated arm and monosomy for the missing arm, with associated
phenotypic consequences.
Ring chromosome
A ring chromosome arises by breakage of
both the short and long arms and subsequent
fusion of the broken ends. Rings may exert
phenotypic effects by two mechanisms. First,
material is lost from the two arms, leading
to monosomy for these regions. Second, the
ring tends to be unstable during cell division:
it may be lost from some cells, leading to
monosomy, while in other cells a sister chromatid exchange within the ring may result in a
double-sized ring and consequent trisomy. As
a result, individuals with ring chromosomes
may have many cell populations with different
constitutions with respect to the ring. Specific
phenotypic effects vary widely with the chromosome involved and amount of chromosome
material lost.
Translocation
Translocation (Fig. 8.1.2) involves the exchange of segments from one chromosome to
another, usually in a reciprocal fashion. In a
balanced reciprocal translocation, breaks occur in each chromosome and the broken segments are exchanged between the two chromosomes so that no material is lost or gained. A
carrier for a balanced reciprocal translocation
may be asymptomatic, although some individuals manifest a phenotype related to disruption or dysregulation of a gene(s) at the site of
breakage. During meiosis, the chromosomes
involved in a balanced reciprocal translocation
pair in a quadrivalent configuration. Depending on how the chromosomes separate at the
first meiotic division, gametes may receive a
normal chromosome complement, one including the balanced translocation chromosomes,
or an unbalanced chromosomal complement.
The latter leads to partial trisomy and monosomy, which may result in miscarriage or multiple congenital anomalies.
The acrocentric chromosomes (13, 14, 15,
21, and 22) can engage in translocations with
one another in which fusions in repetitive sequences in the short arms result in dicentric
chromosomes (referred to as Robertsonian
translocations; Fig. 8.1.2). If a parent who has
a Robertsonian translocation involving chromosome 21 produces a gamete with both the
translocated chromosome and the normal 21,
this may lead to trisomy 21 in the offspring.
Clinical
Cytogenetics
8.1.9
Current Protocols in Human Genetics
Supplement 89
Robertsonian
translocation
reciprocal
translocation
Insertion
This is a form of translocation in which material is deleted from one chromosome and inserted into another. The inserted material can
be in its normal orientation or inverted relative to the centromere. Partial trisomies can result after meiotic segregation of chromosomes
carrying an insertion.
Complex rearrangements
Translocations or insertions can involve
more than two chromosomes. This occurs particularly with acquired chromosome abnormalities such as those observed in tumors
(Korf, 2010).
Mosaicism
The presence of two cell lines where chromosomal anomalies may be found in some but
not all cells of an individual is a condition
referred to as mosaicism. This results from
occurrence of the chromosome change during
embryonic development. The clinical sequelae
depend on the nature of the anomaly and the
proportion and distribution of cells affected.
In some instances, abnormal cell lines may be
confined to extraembryonic membranes. Effects of such mosaicism on fetal well being
are under study (Kalousek et al., 1992).
Overview of
Clinical
Cytogenetics
Uniparental disomy (UPD) is a phenomenon in which both copies of a chromosome are inherited from one parent. In most
cases, this probably results from an initial
nondisjunction event leading to trisomy, and
8.1.10
Supplement 89
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